Functions And Molecular Mechanisms Of MACC1 In Pancreatic Cancer Progression

MACC1(Metastasis-associated in colon cancer-1)was identified in 2008 as an oncogene,the name came from the fact that MACC1 was highly expression in colorectal cancer(CRC)and significantly correlated with tumor metastasis.Emerging evidence has shown that MACC1 is not only upregulated in CRC,but also in other neoplasms.In digestive system tumors,MACC1 are increased in CRC,esophagus cancer,gastric cancer,liver cancer and cholangiocarcinoma,the upregulation of MACC1 is related to aggressive characteristics and served as a risk factor for survival in patients.Both in vitro and in vivo experiments showed that inhibition of MACC1 could suppress tumor progression.Therefore,MACC1 may consider as a therapeutic target.Recently,it has been found that MACC1 was enriched in serum from patients with digestive cancers,also its upregulation was closely related with shorter survival time and advanced tumor stages,which indicated that MACC1 might be a potential cancerous biomarker.However,the functions and mechanisms of MACC1 in pancreatic cancer were remained largely unknown.Here,we explore the role of MACC1,hoping to provide a new target for pancreatic cancer treatment.Objective:To investigate the functions and molecular mechanisms of MACC1 in pancreatic cancer progression in vitro and in vivo.Method:1.Immunohistochemistry(IHC)was performed to measure the expression of MACC1 in tissue array which included 162 primary PDAC and 127 adjacent normal pancreatic tissue specimens.The resected tissues were identified by two experienced pathologists independently(The intensity of the staining was characterized as: negative—0 point,weak detectable—1 point,distinct—2 point and very strong—3 point.The percentage of positive cell was determined as:less than 5%--0 point,6%-25%--1 point,26%-50%--2 point,51%-75%--3 point,76%-100%--4 point).The results were used to analyzed the correlation of MACC1 expression with clinicopathological features and prognosis of patients with pancreatic cancer.2.QRT-PCR(quantitative real time polymerase chain reaction,q RT-PCR)and Western blot were operated to detect the m RNA and protein level in normal pancreatic ductal epithelial cells and PDAC cell lines.4.For the in vitro cell-behavior experiments,CCK-8 assay,cloning formation assay,cell cycle and apoptosis assay were performed to clarify the impacts of MACC1 on proliferative abilities.Wound-healing assay,migration assay,invasion assay were tested to evaluate the effects of MACC1 on migration capacities.5.For the in vivo experiments,the subcutaneous tumor formation model and metastatic models(pulmonary metastatic model and liver metastatic model)in nude mice were carried out to detect the effects of MACC1 on PDAC proliferation and migration abilities.6.After overexpression or knockdown of MACC1,m RNA,protein and phosphorylated protein expression level of c-Met(MET proto-oncogene,receptor tyrosine kinase)were detected by q RT-PCR and Western Blot.7.Cells were treated with or without c-met inhibitor,Western blot was performed to validate the effect of the inhibitor.MACC1 overexpression cells were added with or without c-met inhibitor,then migration and invasion assay were carried out to evaluate the effects on metastatic abilities.8.Tumor metastatic m RNA array combined with GO(Gene Ontology,GO)/KEGG(Kyoto Encyclopedia of Genes and Genomes,KEGG)pathway analysis was used to screen the metastasis correlated downstream genes and pathways.9.QRT-PCR was performed to validate the screened genes(top 10 upregulated genes),FN1(fibronectin 1)was selected as the effect gene through immunoblotting assay.10.The expression level of FN1 in PDAC was analyzed by TCGA(The cancer genome atlas)database.Also,the correlation between MACC1 and FN1 was further calculated.Meanwhile,the protein level of FN1 and MACC1 were detected in 10 paired PDAC and normal tissues.11.Immunofluorescence,Immunohistochemistry and clinical data analysis were operated to further evaluate the correlation between MACC1 and FN1,meanwhile,the clinical significance was assessed.12.Full length of FN1 promoter sequence were constructed into the reporter gene plasmid,Dual-Luciferase reporter assay was used to clarify the effect of MACC1 on FN1 transcription.13.The overexpression and knockdown plasmids targeting FN1 were synthesized then packaged into lentivirus.The MACC1 stably overexpressing cells were infected with FN1 knockdown lentivirus,in addition,the MACC1 decreasing cells were treated with FN1 overexpression lentivirus.After selecting with puromycin,wound-healing assay,transwell migration assay and invasion assay were performed to detect the rescue abilities of FN1.14.MACC1 probable associative proteins,including HSF1(heat shock transcription factor 1),HMGA2(high mobility group AT-hook 2),ATF3(activating transcription factor 3),EGR1(early growth response 1),CREB1(c AMP responsive element binding protein 1,SNAI1(snail family transcriptional repressor 1),LEF1(lymphoid enhancer binding factor 1),were validated by COIP(Co-immunoprecipitation)experiments,finally,SNAI1 was confirmed to interact with MACC1 in different PDAC cells.15.Sh RNA targeting SNAI1 was constructed into plasmid.MACC1 overexpression cells were transfected with sh-SNAI1 or control plasmids,respectively,FN1 expression was detected by q RT-PCR and Western Blot.Results:1.The expression of MACC1 was significantly increased in PDAC tissues compared with adjacent tissues.Meanwhile,the expression level of MACC1 was positively correlated with advanced tumor stage and distant metastasis.In addition,highly MACC1 level was associated with a shorter survival time of PDAC patients.The result of Western blot also verified that MACC1 was increased in PDAC tissues.2.RT-PCR and Western Blot found that both RNA and protein level of MACC1 were upregulated in PDAC cells than normal ductal epithelial cells.3.According to the MACC1 expression level in different cell lines,PANC-1 and SUIT-2 were selected as MACC1 low-expressing cells and were constructed into MACC1-overexpressing stable cells.MACC1 highly expressing cells,such as BXPC-3,was used to construct stable MACC1-downregulated cells.4.The migration abilities of PDAC cells were significantly inhibited in MACC1 downregulated cells,on the contrary,upregulation of MACC1 could promote the aggressive behaviors of PDAC cells.5.MACC1 promoted the metastasis of PDAC cells in vivo.6.RT-PCR and Western Blot experiments showed that decreased MACC1 did not affect the m RNA,protein and phosphorylated protein level of c-Met.7.The c-Met inhibitor,JNJ38877605,could not rescue the tumor-promoting phenotypes of MACC1.8.Tumor metastatic RNA-seq and GO/KEGG pathway analysis found that MACC1 mainly affected signal transduction,protein interaction and proliferation abilities of PDAC cells.Meanwhile,upregulated MACC1 could increase a series of oncogenes,such as FN1(fibronectin 1),PTGS1(prostaglandin-endoperoxide synthase 2),MMP10(matrix metallopeptidase 10),SYK(spleen associated tyrosine kinase),TIMP4(TIMP metallopeptidase inhibitor 4),CHD1(cadherin1),FLT1(fms related receptor tyrosine kinase 1),TIMP3(TIMP metallopeptidase inhibitor 3),SPARC(secreted protein acidic and cysteine rich)and TFF1(trefoil factor 1).9.MACC1 could increase the transcriptional levels of FN1,PTGS2 and MMP10,furthermore,immunoblot assay verified that FN1 was the downstream molecule of MACC1.10.TCGA database revealed that FN1 was overexpressed in PDAC tissues than normal tissues,in addition,Pearson correlation analysis showed that the expression level of FN1 was positively correlated with MACC1.Western blot results were consistent with the TCGA database: FN1 and MACC1 both highly expressed in PDAC tissues.11.Immunofluorescence further confirmed that MACC1 could increase the expression of FN1.What`s more,immunohistochemical experiments in tissue array validated that FN1 was highly expressed in PDAC tissues compared with adjacent tissues,and positively correlated with poor prognosis.The shortest survival time was observed in the groups with high expression of both FN1 and MACC1.12.Dual-Luciferase reporter assay revealed that MACC1 enhanced the activity of FN1 Promoter.13.Reduction of FN1 attenuated MACC1-facilitated migration and invasion in PDAC cell lines,and overexpression of FN1 increased the promotion of migration and invasion abilities of MACC1.14.CO-IP found that HMGA2 and SNAI1 were interacted with MACC1.After validating in different cell lines,SNAI1,but not HMGA2,was confirmed to bind to MACC1.15.Knockdown of SNAI1 attenuated MACC1-mediated FN1 expression.Conclusion:Our findings reveal that MACC1 can interact with SNAI1,thereby enhancing the transcriptional activity of FN1 and increasing the expression level of FN1,consequently facilitates the metastasis of pancreatic cancer cells.